The overall goal of this research is to develop a mechanistic understanding of the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. Genetic analysis has led to the identification of a very large number of components (Vps proteins) required along the vacuolar assembly pathway in yeast, and the major focus of this proposal is a functional characterization of Vps-dependent membrane transport to and from the vacuole. A complex of proteins has been identified that function in retrograde membrane transport from the prevacuole/ endosome back to the Golgi complex, and this complex will be investigated further both genetically and biochemically. The function of the yeast post-Golgi SNARE (both v-SNAREs and t- SNAREs) proteins will be investigated in greater detail to elucidate the molecular mechanistic basis for the specificity of the large number of membrane fusion steps that occur throughout the vacuolar network of membranes. Focus will also be on the newly discovered retrograde membrane transport pathway out of the vacuole, a pathway by which membrane proteins are transported back to the prevacuole/endosome. Combining genetic and biochemical approaches, the components of the relevant protein complexes will be identified, and their precise execution points and requirements in the three major biogenesis pathways will be investigated. Studies of membrane traffic in the yeast Saccharomyces cerevisiae have proven tremendously useful to a broader understanding of membrane transport in all eukaryotic cells because of the remarkable similarity in mechanisms and proteins that regulate these processes from yeast to humans. It has become clear in the last five years that virtually every Vps gene/protein discovered by us and others in yeast has its mammalian homologue, and many of these have been shown to function in yeast. These basic mechanistic studies in yeast should provide important insights into our understanding of many lysosomal storage diseases, as well as likely defects in post-Golgi pigment granule function in syndromes such as Chediak-Higashi and Hermansky-Pudlak.